The last three decades has seen considerable effort in the development of improved methods for the isolation and purification of nucleic acids from biological sources. This has been due mainly to the increasing applications of nucleic acids in the medical and biological sciences. Genomic DNA isolated from blood, tissue or cultured cells has several applications, which include PCR, sequencing, genotyping, hybridization and southern blotting. Plasmid DNA has been utilized in sequencing, PCR, in the development of vaccines and in gene therapy. Isolated RNA has a variety of downstream applications, including blot hybridization, in vitro translation, cDNA synthesis and RT-PCR.
The analysis and in vitro manipulation of nucleic acids is typically preceded by a nucleic acid isolation step in order to free the nucleic acid from unwanted contaminants which may interfere with subsequent processing procedures. For the vast majority of procedures in both research and diagnostic molecular biology, extracted nucleic acids are required as the first step. In a typical DNA extraction protocol, cells or homogenized tissue samples containing the nucleic acid of interest are harvested and lysed using standard methods, for example using enzymes such as Proteinase K and lysozyme; detergents, such as SDS, Brij, Triton X100, or using other chemicals such as sodium hydroxide, guanidium isothiocyanate, etc. (See for example, Sambrook et al, Molecular Cloning—A Laboratory Manual 2nd edition 9.14 (New York: Cold Spring Harbor Laboratory 1989). Following removal of the cellular debris, the crude lysate is treated with organic solvents such as phenol/chloroform to extract proteins. RNA may be removed or reduced if required by treatment of the enzymes such as RNAse. However, the presence of contaminants such as salts, phenol, detergents and the like can interfere with many downstream manipulations for which the nucleic acid is intended.
Currently several procedures are available for the chromatographic purification of DNA (genomic and plasmid) and RNA, for example, by employing silica based membrane purification, size exclusion chromatography, reversed phase chromatography, gel filtration, magnetic bead based purification, or ion-exchange chromatography. Ion exchange chromatography is one of the most commonly used separation and purification methods and has been used for purification of plasmid DNA, genomic DNA and RNA.
See for example, U.S. Pat. No. 6,410,274 (Bhikhabhai), U.S. Pat. No. 6,310,199 (Smith et al), U.S. Pat. No. 6,090,288 (Berlund et al), U.S. Pat. No. 5,990,301 (Colpan et al), U.S. Pat. No. 5,856,192, U.S. Pat. No. 5,866,428 (Bloch), U.S. Pat. No. 5,801,237 (Johansson), EP 1125943 b1 (Macherey-Nagel GmbH & Co), EP 992583 B1, EP 616639 (Qiagen), U.S. Pat. No. 5,707,812, U.S. Pat. No. 5,561,064 (Vical Inc.).
While anion exchange chromatographic procedures for the purification of nucleic acids have been extensively referenced, one of the shortcomings of current protocols is the impaired recovery of nucleic acid during the elution step, (Endres, H. N. et al, Biotechnol. Appl. Biochem., (2003), 37(3), 259-66; Prazeres, D. M. et al, J. Chromatog. A. (1998), 806(1), 31-45; Urthaler J. et al, Acta Biochim. Pol., (2005), 52(3), 703-11; Ferreira, G. N. et al, Bioseparation, (2000), 9(1), 1-6.; Ferreira, G. N., et al, Biotechnol. Prog., (2000), 16(3), 416-24. Addition of organic agents such as polyols and alcohols during adsorption and desorption has been shown to improve selectivity and recovery during anion exchange purification of DNA (Tseng, W. C. et al, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., (2003), 791(1-2), 263-72). However, there appear to be no reports that specifically address the recovery issues often seen during DNA desorption from anion exchange resins. The present invention addresses this problem since it relates to improving recoveries of bound DNA from anion exchange resins. In particular, the invention allows improved desorption of the DNA from the solid support without further manipulation of the protocol.
Plasmid DNA, genomic DNA and RNA have similar charge properties to one another and are polyanions having high charge density. Binding to positively charged ion exchange resins is therefore possible in the presence of up to 0.7M sodium chloride, depending on the length and conformation of the nucleic acid to be adsorbed. An increase in nucleic acid length as well as double stranded conformation results in an increase in binding strength between the nucleic acid and the anion exchanger. However, this effect is only proportional to nucleic acid length up to about 2 kilobases. The very strong interaction between the negatively charged phosphate backbone of the nucleic acid and ion exchange resin hampers elution of the nucleic acid using conventional methods, where a simple increase in ionic strength of the salt eluant is sufficient for recovery of 70-100% of the bound material. However, in the case of longer chain nucleic acids, an increase in ionic strength up to 3M salt only allows recoveries of 20-50% of the bound nucleic acid. Recovery of the remaining bound material can be accomplished with a combination of high salt concentration and elevated pH using sodium hydroxide. However, sodium hydroxide is not only caustic, but may also lead to irreversible denaturation of nucleic acids and degradation over time.
Currently, ion-exchange resin is either provided in dried format (silica based resins) or in water in the presence of antimicrobial growth inhibitors. The resin is equilibrated with sample loading solution when the purification protocol is carried out. It is advantageous to simplify the process by eliminate this resin equilibration step.